Pneumonias resulting from failure to eradicate bacterial (Streptococcus pneumonia, Haemophilus influenza, Staphylococcus aureus, Pseudomonas aeruginosa) and viral (rhinovirus, influenza virus) infections are responsible for a tremendous burden of hospitalizations and deaths. Using mouse models, we established that the protease inhibitor SerpinB1, an ancient nucleocytoplasmic protein, (i) protects anti-microbial capacity during P. aeruginosa and influenza virus infections and (ii) acts as a critical negative regulator of NETosis. NETosis (Neutrophil Extracellular Trap production) is a programmed death pathway induced by pathogens and inflammatory mediators in which dying neutrophils extrude linearized DNA coated with potent antimicrobial enzymes: histones, myeloperoxidase and neutrophil serine proteases (NSPs). Although NETosis can be protective by preventing dissemination of infection, this death pathway is frequently pathological because excess NETs inflict serious damage to host tissue and destroy anti-microbial defenses in pneumonia and other inflammatory and autoimmune diseases. To examine the mechanisms by which SerpinB1 inhibits NETosis, we will identify and characterize the required proteases that co-function with SerpinB1. We will determine in Aim 1 whether NETs are produced by neutrophils that are singly, doubly and triply deleted for elastase, cathepsin-G or proteinase 3 (granule serine proteases inhibited by SerpinB1 and collectively known as NSPs) and by wild-type (WT) neutrophils treated with inhibitors and will verify the findings in vivo. We will determine whether the require NSP is located on the cell surface by the use of non-permeable serine protease inhibitors. The second aim builds on our recent findings that SerpinB1 migrates into the nucleus early during NETosis where it inhibits the activity (or activation) of PAD4 (peptidylarginine deiminase-4), the essential enzyme that deiminates (citrullinates) histone tail arginine residues, causing chromatin decondensation, expansion of the nucleus, and extrusion of NETs. To dissect the mechanism of this restriction, we will determine in Aim 2 whether (i) PAD4 protein differs (in amount and molecular form) between WT and serpinb1-/- neutrophils and whether (ii) SerpinB1 inhibits PAD4 activation in a broken cell nuclear preparation from serpinb1-/- neutrophils. The final aim will determine whether nuclear cysteinyl cathepsins might co-function in regulating NETosis based on the findings that these proteases are inhibited by SerpinB1 and localize to the nucleus in a number of cells. We will determine in Aim 3 whether (i) cysteinyl cathepsins are expressed in the nucleus of murine neutrophils, either constitutively or during NETosis (by the use of class-specific mechanism-based probes) and whether (ii) cathepsin inhibitors block NETs production. The proposed studies will define key elements of the mechanism of NET production and potentially identify targets for therapy to dampen excess NETosis and thereby decrease inflammatory injury and protect anti-microbial defense in pneumonia and other inflammatory diseases.